Cytokeratin 8 protects from hepatotoxicity, and its ratio to cytokeratin 18 determines the ability of hepatocytes to form Mallory bodies.

Department of Pathology, University of Graz, Graz, Austria. kurt.zatloukal@kfunigraz.ac.at

Abstract

In alcoholic hepatitis, a severe form of alcohol-induced toxic liver injury, as well as in experimental intoxication of mice with the porphyrinogenic drugs griseofulvin and 3,5-diethoxycarbonyl-1, 4-dihydrocollidine, hepatocytes form cytoplasmic protein aggregates (Mallory bodies; MBs) containing cytokeratins (CKs) and non-CK components. Here we report that mice lacking the CK8 gene and hence CK intermediate filaments in hepatocytes, but still expressing the type I partner, ie, the CK18 gene, do not form MBs but suffer from extensive porphyria and progressive toxic liver damage, leading to the death of a considerable number of animals (7 of 12 during 12 weeks of intoxication). Our observations show that 1) in the absence of CK8 as well as in the situation of a relative excess of CK18 over CK8 no MBs are formed; 2) the loss of CK8 is not compensated by other type II CKs; and 3) porphyria and toxic liver damage are drastically enhanced in the absence of CK8. Our results point to a protective role of CKs in certain types of toxic liver injury and suggest that MBs by themselves are not harmful to hepatocytes but may be considered as a product of a novel defense mechanism in hepatocytes.

Graphic presentation showing the increased lethality of CK8 knockout mice with increasing time of DDC intoxication. Wild-type (wt) (CK8+/+), heterozygous (CK8+/−), and homozygous CK8 knockout mice (CK8−/−) were fed a diet containing 0.1% DDC. No wt mice (0 of 13) died within an intoxication period of 12 weeks, whereas 2 of 11 of the CK8+/− mice and seven of 12 of the CK8−/− mice died.

Histogram presenting porphyrin concentrations in liver. Liver tissue was obtained from wt mice fed a standard diet and from CK8 knockout mice as well as from transgenic mice expressing the neomycin resistance gene after 3 months (3 months) of DDC intoxication. Total porphyrin content in the liver was measured fluorometrically after acid extraction. Porphyrin concentration was calculated per gram of liver tissue. Bars represent mean values (with standard deviations) from five different mice per group (CK8+/+, CK8+/−, CK8−/−) and, for control, three transgenic mice containing the neomycin resistance gene under the control of the thymidine kinase promoter (M-TKneo). Note the significant increase of liver porphyrin in CK8+/− and CK8−/− mice, with the highest liver porphyrin accumulation in the latter. The neomycin resistance gene itself, which is present in the CK8 knockout as well as M-TKneo mice, had no influence on the DDC-induced porphyria. Protoporphyrin is not detected (n.d.) in normal mouse liver (CO).

Double-label immunofluorescence microscopy of livers from 12-week DDC-intoxicated mice showing that MBs do not form in the absence of CK8. A–H: CK8 and CK18 were immunostained with rabbit antibodies (50K160, red). A–C, G, H: The mAb MM120–1 (green) also reacts with MBs (yellow, due to co-localization with CK) found only in wt as well as M-TKneo mice; D–F: mAb SMI 31 (green) recognizes a 62- to 65-kd MB component which co-localizes with CK in MBs. A and D, wt mice; B and E, CK8+/− mice; C and F, CK8−/− mice; G, liver of a M-TKneo mouse fed a standard diet; H, liver of a M-TKneo mouse fed DDC for 12 weeks. Note that the presence of the neomycin resistance gene in M-TKneo mice has no effect on DDC-induced derangement of the CK IF cytoskeleton and MB formation. Scale bar, 20 μm.